tuning-degradation-to-achieve-specific-and-efficient-protein-depletion

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

University of Edinburgh

University of Oxford

It is widely accepted that heterochromatin is necessary to maintain genomic stability. However, direct experimental evidence supporting this is slim. Previous studies using either enzyme inhibitors, gene knockout or knockdown studies all are subject to the caveat that drugs may have off-target effects and enzymes that modify chromatin proteins to support heterochromatin formation may also have numerous other cellular targets as well. Here, we describe PREditOR (protein reading and editing of residues), a synthetic biology approach that allows us to directly remove heterochromatin from cells without either drugs or global interference with gene function. We find that removal of heterochromatin perturbs mitotic progression and causes a dramatic increase in chromosome segregation defects, possibly as a result of interfering with the normal centromeric localization of the chromosomal passenger complex.

PREditOR: a synthetic biology approach to removing heterochromatin from cells.

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the β-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and β-estradiol systems results in a tightly controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of β-estradiol preincubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.

A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast.

There is increasing evidence from yeast to humans that pre-mRNA splicing occurs mainly co-transcriptionally, such that splicing and transcription are functionally coupled. Currently, there is little insight into the contribution of the core transcription elongation machinery to co-transcriptional spliceosome assembly and pre-mRNA splicing. Spt5 is a member of the core transcription elongation machinery and an essential protein, whose absence in budding yeast causes defects in pre-mRNA splicing. To determine how Spt5 affects pre-mRNA splicing, we used the auxin-inducible degron system to conditionally deplete Spt5 in  and assayed effects on co-transcriptional spliceosome assembly and splicing. We show that Spt5 is needed for efficient splicing and for the accumulation of U5 snRNPs at intron-containing genes, and therefore for stable co-transcriptional assembly of spliceosomes. The defect in co-transcriptional spliceosome assembly can explain the relatively mild splicing defect as being a consequence of the failure of co-transcriptional splicing. Co-immunoprecipitation of Spt5 with core spliceosomal proteins and all spliceosomal snRNAs suggests a model whereby Spt5 promotes co-transcriptional pre-mRNA splicing by stabilising the association of U5 snRNP with spliceosome complexes as they assemble on the nascent transcript. If this phenomenon is conserved in higher eukaryotes, it has potential to be important for co-transcriptional regulation of alternative splicing.

Isabella E. Maudlin

Wellcome Centre for Cell Biology,
School of Biological Sciences,
Wellcome Centre for Cell Biology, School of Biological Sciences

University of Edinburgh

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

Isabella E. Maudlin

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University of Edinburgh

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

Wellcome Centre for Cell Biology,
School of Biological Sciences,
Wellcome Centre for Cell Biology, School of Biological Sciences